白藜芦醇调控内皮细胞凋亡及促血栓分子P-Selectin、vWF表达的实验研究
发布时间：2019-06-28 20:29
【摘要】：DVT是骨科大手术后常见的临床并发症,属于静脉回流障碍性疾病,发病率高,危害性大,是骨科手术患者围手术期死亡的重要原因之一。DVT发病机制复杂,涉及内皮细胞、血小板、白细胞等多方面因素,近年来研究表明,血管内皮细胞损伤,是静脉血栓形成最重要的原因之一,内皮细胞损伤后可释放各种粘附分子、血小板活化因子、组织因子,增强凝血,促进DVT形成;然而,氧化应激反应、炎症反应是引起静脉内皮细胞损伤的常见原因,氧化应激、炎症可引起机体活性氧、炎症因子产生增加,使细胞脂质过氧化、NO减少,从而引起内皮细胞损伤、凋亡,促进DVT形成;同时,有研究表明,NF-κB可以通过上调组织因子表达,促进凝血,从而影响DVT形成;许多研究表明,白藜芦醇具有抗氧化、抗炎、保护内皮细胞损伤的功能,但是,在DVT形成方面却罕见报道。因此,本研究旨在探讨:白藜芦醇对内皮细胞氧化损伤、凋亡的抑制作用,以及对促血栓分子P-Selectin、vWF表达的影响及调控机制。[目的]1.探讨白藜芦醇对内皮细胞氧化损伤、凋亡的影响。2.探讨白藜芦醇对内皮细胞损伤后促血栓分子P-Selectin、vWF表达的影响及调控机制。[方法]本研究分为两部分:1.第一部分,本实验部分分为三组:(1)空白对照组;(2)H202组:以20μmo/L的H202处理人脐静脉内皮细胞24小时建立静脉内皮细胞损伤模型;(3)RES+H2O2组:以30μmo/L的白藜芦醇预处理HUVECs 2小时后,再以200μmo/L的H202处理细胞24小时。采用MTT法检测各组细胞活力,以探讨白藜芦醇对H202诱导的HUVECs损伤的保护作用;采用荧光探针DCFH-DA捕获法、激光共聚焦显微镜检测细胞内活性氧ROS含量,以探讨白藜芦醇对内皮细胞氧化应激的影响;Hoechst 33258染色法、荧光显微镜,AnnexinV-FITC/PI双染法、流式细胞仪检测细胞凋亡,以探讨白藜芦醇对内皮细胞凋亡的影响。2.第二部分,本实验部分分为四组;(1)空白对照组;(2)H202组:本组与第一部分相同;(3)RES+H2O2组:本组与第一部分相同;(4)BAY11-7082+H2O2组:以5μmol/LNF-κB特异性抑制剂BAY 11-7082预处理细胞4小时后,再加入 200μmol/L H2O2 孵育细胞 24 小时。采用 Real-Time PCR、western blot检测各组NF-κB、P-Selectin、vWF基因及蛋白表达变化,以探讨白藜芦醇对内皮细胞损伤后促血栓分子P-Selectin、vWF表达的影响及调控机制。[结果]1.第一部分实验结果:采用200μmol/L的H2O2处理HUVECs 24小时,即H202组,与对照组相比,细胞活力明显降低(P0.01),细胞内ROS含量明显增多(P0.01),细胞凋亡率明显增加(P0.01);然而采用30μmo/L白藜芦醇预处理HUVECs 2小时后,再用200μmo/L H202孵育细胞24小时,即RES+H2O2组,与H202组相比,细胞活力明显增高(P0.01),细胞内ROS含量明显减少(P0.01),细胞凋亡率明显减少(P0.01)。2.第二部分实验结果:(1)用200μmol/L H202处理HUVECs 24小时后,即H202组,其NF-κB、P-Selectin、vWFmRNA及蛋白表达较对照组明显增高(P0.01)。(2)然而在加入了 NF-κB的特异性抑制剂BAY 11-7082 5μmol/L预处理4小时后,再用200μmol/LH2O2处理细胞24小时,即BAY11-7082+H2O2组,与H2O2组比较,其NF-κBmRNA及蛋白表达出现了下调(P0.01),P-Selectin、vWFmRNA及蛋白表达也随之下调(P0.05)。(3)而在加入30μmol/L的白藜芦醇预处理2小时后,再用200μmol/LH2O2处理细胞24小时,即RES+H2O2组,与H2O2组比较,其NF-κBmRNA及蛋白表达出现了下调(P0.01),P-Selectin、vWFmRNA及蛋白表达也随之下调(P0.05)。(4)RES+H2O2组与 BAY 11-7082+H2O2组比较,BAY 11-7082+H2O2组NF-κB mRNA及蛋白表达下调更明显(P0.01),vWFmRNA及蛋白表达下调也更明显(P0.01)。[结论]1.白藜芦醇可减少内皮细胞损伤、凋亡。2.白藜芦醇可减少内皮细胞损伤后ROS生成。3.NF-κB参与了内皮细胞损伤后促血栓分子P-Selectin、vWF表达。4.白藜芦醇可以抑制内皮细胞损伤后促血栓分子P-Selectin、vWF的激活,且可能通过NF-κB信号通路发挥效应;推测其对DVT形成具有一定的防治作用。
[Abstract]:DVT is a common clinical complication after large-size orthopedic surgery, and it is one of the important causes of the perioperative death in the patients with venous reflux disease, high incidence and high risk. The pathogenesis of the DVT is complicated, and it is related to the factors such as endothelial cell, platelet, and white blood cell. In recent years, the research has shown that the damage of the vascular endothelial cells is one of the most important reasons for venous thrombosis, and the endothelial cells can release various adhesion molecules and platelet activating factors after the endothelial cells are damaged. the tissue factor, the enhanced blood coagulation and the promotion of the formation of the DVT; however, the oxidative stress reaction, the inflammatory reaction is a common cause of the damage of the vein endothelial cells, the oxidative stress and the inflammation can cause the active oxygen and the inflammation factor of the organism to increase, and the lipid peroxidation and the NO of the cells are reduced, so as to cause injury and apoptosis of the endothelial cells and promote the formation of the DVT; at the same time, the research shows that the NF-B can increase the expression of the tissue factor and promote the coagulation, thereby affecting the formation of the DVT, The function of protecting endothelial cell damage, however, is rare in the formation of DVT. Therefore, the purpose of this study is to study the effect of the white and aloe on the oxidative damage and apoptosis of the endothelial cells, as well as the effect of the expression of P-Selectin and vWF on the thrombogenic molecules. [Objective] 1. Objective To study the effect of the white-and-white aloe on the oxidative damage and apoptosis of the endothelial cells. To study the effect and regulation mechanism of the expression of P-Selectin and vWF on the endothelial cells after the injury of the endothelial cells. [Method] This study is divided into two parts:1. The first part, this experiment part was divided into three groups: (1) blank control group; (2) H202 group: human umbilical vein endothelial cells were treated with 20. mu. mo/ L H202 for 24 hours to establish a model of vein endothelial cell damage; (3) RES + H2O2 group: after the HUVECs were pre-treated with 30. m The cells were treated with 200. m u.M/ L of H202 for 24 hours. The activity of the cells in each group was detected by MTT method, and the protective effect of the white and aloe on the H202-induced HUVECs injury was discussed. The content of reactive oxygen ROS in the cells was detected by using the fluorescence probe DCFH-DA capture method and the laser confocal microscope. Hoechst 33258 staining, fluorescence microscope, Annexin V-FITC/ PI double staining method and flow cytometry were used to detect the apoptosis of the cells. The second part, this experiment part is divided into four groups; (1) blank control group; (2) H202 group: this group is the same as the first part; (3) RES + H2O2 group: this group is the same as the first part; (4) BAY11-7082 + H2O2 group: after 4 hours of pretreatment of BAY 11-7082 with 5. m The cells were incubated with 200. m u.mol/ L of H2O2 for 24 hours. Using Real-Time PCR and western blot, the expression of NF-B, P-Selectin, vWF and protein in each group were detected, and the effect and regulation mechanism of the expression of P-Selectin and vWF on the endothelial cells after the injury of the endothelial cells were discussed. [Results] 1. The results of the first part showed that the cell viability of HUVECs was significantly lower than that of the control group (P0.01). The content of ROS in the cells increased significantly (P0.01), and the rate of apoptosis was significantly increased (P0.01). However, after the pretreatment of HUVECs by 30 & mu; mo/ L, the cells were incubated with 200 & mu; mo/ L H202 for 24 hours, that is, the RES + H2O2 group, and the activity of the cells was significantly higher than that of the H202 group (P0.01). The content of ROS in the cells was significantly decreased (P0.01), and the apoptosis rate of the cells was significantly decreased (P0.01). The results of the second part: (1) After the treatment of HUVECs for 24 hours with 200 & mu; mol/ L H202, the expression of NF-B, P-Selectin, vWFmRNA and protein in the H202 group was significantly higher than that in the control group (P0.01). (2) After the pretreatment of BAY 11-7082 5umol/ L, the specific inhibitor BAY 11-7082 5 & mu; mol/ L was treated with 200 & mu; mol/ L H _ 2O _ 2 for 24 hours, that is, BAY11-7082 + H2O2 group, and the expression of NF-BmRNA and protein of BAY11-7082 + H2O2 group was down-regulated (P0.01), P-Selectin, The expression of vWFmRNA and protein was also down-regulated (P0.05). (3) After the pre-treatment of 30. m u.mol/ L of Phragon for 2 hours, the cells were treated with 200. m u.mol/ L H _ 2O _ 2 for 24 hours, that is, the RES + H2O2 group, and the expression of NF-BmRNA and protein in the group was down-regulated (P0.01), and the expression of P-Selectin, vWFmRNA and protein was also down-regulated (P0.05). (4) Compared with BAY 11-7082 + H2O2 group, the expression of NF-EMAB mRNA and protein in BAY 11-7082 + H2O2 group was lower than that of BAY 11-7082 + H2O2 group (P0.01). [Conclusion] 1. Phragon can reduce the damage and apoptosis of the endothelial cells. 3. NF-EMAB was involved in the expression of P-Selectin and vWF after endothelial cell injury. It can inhibit the activation of P-Selectin and vWF, and may play an important role in the formation of DVT.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68
本文编号：2507590
[Abstract]:DVT is a common clinical complication after large-size orthopedic surgery, and it is one of the important causes of the perioperative death in the patients with venous reflux disease, high incidence and high risk. The pathogenesis of the DVT is complicated, and it is related to the factors such as endothelial cell, platelet, and white blood cell. In recent years, the research has shown that the damage of the vascular endothelial cells is one of the most important reasons for venous thrombosis, and the endothelial cells can release various adhesion molecules and platelet activating factors after the endothelial cells are damaged. the tissue factor, the enhanced blood coagulation and the promotion of the formation of the DVT; however, the oxidative stress reaction, the inflammatory reaction is a common cause of the damage of the vein endothelial cells, the oxidative stress and the inflammation can cause the active oxygen and the inflammation factor of the organism to increase, and the lipid peroxidation and the NO of the cells are reduced, so as to cause injury and apoptosis of the endothelial cells and promote the formation of the DVT; at the same time, the research shows that the NF-B can increase the expression of the tissue factor and promote the coagulation, thereby affecting the formation of the DVT, The function of protecting endothelial cell damage, however, is rare in the formation of DVT. Therefore, the purpose of this study is to study the effect of the white and aloe on the oxidative damage and apoptosis of the endothelial cells, as well as the effect of the expression of P-Selectin and vWF on the thrombogenic molecules. [Objective] 1. Objective To study the effect of the white-and-white aloe on the oxidative damage and apoptosis of the endothelial cells. To study the effect and regulation mechanism of the expression of P-Selectin and vWF on the endothelial cells after the injury of the endothelial cells. [Method] This study is divided into two parts:1. The first part, this experiment part was divided into three groups: (1) blank control group; (2) H202 group: human umbilical vein endothelial cells were treated with 20. mu. mo/ L H202 for 24 hours to establish a model of vein endothelial cell damage; (3) RES + H2O2 group: after the HUVECs were pre-treated with 30. m The cells were treated with 200. m u.M/ L of H202 for 24 hours. The activity of the cells in each group was detected by MTT method, and the protective effect of the white and aloe on the H202-induced HUVECs injury was discussed. The content of reactive oxygen ROS in the cells was detected by using the fluorescence probe DCFH-DA capture method and the laser confocal microscope. Hoechst 33258 staining, fluorescence microscope, Annexin V-FITC/ PI double staining method and flow cytometry were used to detect the apoptosis of the cells. The second part, this experiment part is divided into four groups; (1) blank control group; (2) H202 group: this group is the same as the first part; (3) RES + H2O2 group: this group is the same as the first part; (4) BAY11-7082 + H2O2 group: after 4 hours of pretreatment of BAY 11-7082 with 5. m The cells were incubated with 200. m u.mol/ L of H2O2 for 24 hours. Using Real-Time PCR and western blot, the expression of NF-B, P-Selectin, vWF and protein in each group were detected, and the effect and regulation mechanism of the expression of P-Selectin and vWF on the endothelial cells after the injury of the endothelial cells were discussed. [Results] 1. The results of the first part showed that the cell viability of HUVECs was significantly lower than that of the control group (P0.01). The content of ROS in the cells increased significantly (P0.01), and the rate of apoptosis was significantly increased (P0.01). However, after the pretreatment of HUVECs by 30 & mu; mo/ L, the cells were incubated with 200 & mu; mo/ L H202 for 24 hours, that is, the RES + H2O2 group, and the activity of the cells was significantly higher than that of the H202 group (P0.01). The content of ROS in the cells was significantly decreased (P0.01), and the apoptosis rate of the cells was significantly decreased (P0.01). The results of the second part: (1) After the treatment of HUVECs for 24 hours with 200 & mu; mol/ L H202, the expression of NF-B, P-Selectin, vWFmRNA and protein in the H202 group was significantly higher than that in the control group (P0.01). (2) After the pretreatment of BAY 11-7082 5umol/ L, the specific inhibitor BAY 11-7082 5 & mu; mol/ L was treated with 200 & mu; mol/ L H _ 2O _ 2 for 24 hours, that is, BAY11-7082 + H2O2 group, and the expression of NF-BmRNA and protein of BAY11-7082 + H2O2 group was down-regulated (P0.01), P-Selectin, The expression of vWFmRNA and protein was also down-regulated (P0.05). (3) After the pre-treatment of 30. m u.mol/ L of Phragon for 2 hours, the cells were treated with 200. m u.mol/ L H _ 2O _ 2 for 24 hours, that is, the RES + H2O2 group, and the expression of NF-BmRNA and protein in the group was down-regulated (P0.01), and the expression of P-Selectin, vWFmRNA and protein was also down-regulated (P0.05). (4) Compared with BAY 11-7082 + H2O2 group, the expression of NF-EMAB mRNA and protein in BAY 11-7082 + H2O2 group was lower than that of BAY 11-7082 + H2O2 group (P0.01). [Conclusion] 1. Phragon can reduce the damage and apoptosis of the endothelial cells. 3. NF-EMAB was involved in the expression of P-Selectin and vWF after endothelial cell injury. It can inhibit the activation of P-Selectin and vWF, and may play an important role in the formation of DVT.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68
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